The Nobel-price winning method of reprogramming somatic cells into induced pluripotent stem cells by ectopic expression of four key developmental genes c-myc, Sox2, Oct4 and Klf4 is a powerful tool to obtain patient-specific pluripotent cells capable of differentiating into any cell type (Takahashi and Yamanaka, 2006). More recently, different studies have reported that cell type-specific genes can also directly convert one somatic cell type into another, a process reported as cell conversion or transdifferentiation (Vierbuchen and Wernig, 2011). This approach represents a promising strategy to obtain various defined cells for clinics and research, avoiding the potentially tumorigenic pluripotent stage.
A major drawback of known cell conversion or reprogramming strategies is the requirement of introducing ectopic genes which might have undesired effects in later applications of the converted cells. To allow for prolonged and stable expression, the ectopic genes often have to be stably integrated into the genome. Despite the possibilities to tightly control ectopic gene expression, the integrated genes may have undesired effects. For example the introduction of the proto-oncogene c-myc increased the risk of tumor formation in chimeric animals due to c-myc reactivation (Okita et al., 2007). In general, the complex regulation machinery of the genome makes it rather difficult to predict long-term effects of genetic modifications.
To avoid the disadvantages of gene integration in reprogramming approaches, novel methods replace key reprogramming genes by small molecules. Li et al generated IPS cell by replacing certain genes with small molecules that modify specific signaling pathways like Wnt or TGF-β (Li et al., 2012). Yield and efficiency of gene-mediated conversion of fibroblasts into neurons was greatly increased by small molecule based inhibition of BMP, TGF β and GSK3b signaling (Ladewig et al., 2012). In line with these results, small molecules have also been proven valuable tools for directing the differentiation of stem cells. For example, inhibition of bone morphogenetic protein (BMP) and TGF-β signaling leads to highly efficient neural induction of stem cells (Chambers et al., 2009).
The mechanisms by which small molecules influence cell fate decisions are not completely understood. While small molecules can be sufficient to induce differentiation, induction of cell conversion to pluripotency or another somatic cell type still requires ectopic gene expression. This may be due to the fact that changing a phenotype of a terminally differentiated cell into a different cell type is a non-physiological procedure compared to the differentiation of a stem cell into a somatic cell.
Provided herein is a novel method to convert somatic cells into multipotent neural crest cells, which is solely based on small molecule treatment and defined culture conditions and does not require to genetically modify the cells by the introduction of genes.
This novel method employs a novel multikinase inhibitor to transdifferentiate somatic cells into a proliferative neural crest like stage. The neural crest cells can be differentiated into multiple cell types like Schwann cells, chondrocytes, smooth muscle cells or adipocytes.
For example the differentiation of the neural crest cells into Schwann cells can be induced by a specific medium in combination with small molecule based inhibition of defined signaling pathways. The neural mature Schwann cells represent the glia cells of the peripheral nervous system (PNS). Schwann cells fulfill numerous functions like immunoprotection, nutrient supply and myelination of the neurons. Schwann cell dysfunction is the cause for many neurological disorders of the peripheral nervous system, e.g. multiple sclerosis or myelination diseases.
Importantly, this novel cell conversion method does not require the expression of any ectopic gene, but is solely based on chemical treatment. It therefore represents a promising approach to generate patient specific neural crest cells or differentiated cells like Schwann cells, chondrocytes, smooth muscle cells or adipocytes.